Immunoassays represent powerful tools to identify a very wide range of compounds, such as antigens and antibodies. Examples of immunoassays are ELISA (enzyme linked immunosorbent assay), EIA (enzyme immunoassay), and RIA (radio immunoassay). Common to all these immunoassays, is that detection sensitivity is limited by the affinity of typical antibodies.
With the prior art immunoassays, detection is not possible below a certain number of molecules, because the background, i.e. unspecifically bound material, interferes with the results. Detection of very low numbers of antigen is becoming increasingly important, especially for diagnostic applications. Therefore, further developments in sensitivity as well as specificity of immunological assays are desired.
Cantor et al, Science, Vol. 258, 2 October 1992, have previously reported the attachment of oligonucleotides to antibodies in order to permit detection of such antibodies having bound antigen in immune reactions. A streptavidin-protein A chimera that posseses tight and specific binding affinity for both biotin and immunoglobulin G was used to attach biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobilized on microtiter plate wells. Then, a segment of the attached DNA was amplified by PCR (Polymerase Chain Reaction). Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed detection of 580 antigen molecules (9.6×10−22 moles) which is a significant improvement compared to, for example, conventional ELISA.
However, in Cantor et al., the labeled DNA-antibody complexes are assembled in situ during the assay. This can create variable stoichiometry in the assembly of the components and in the attachment of the DNA label. Moreover, extra steps are required for addition of biotinylated reagents and binding proteins. Numerous wash steps are also needed to remove excess reagents and to free assay components of non-specifically bound reagents.
Hendrickson et al., Nucleic Acids Research, 1995, Vol 23, No.3, report an advancement of the Cantor et al. assay that reduces complexity. This is achieved through labeling antibody with DNA by direct covalent linkage of the DNA to the antibody. In this approach, the analyte specific antibody and the 5′ amino modified DNA oligonucleotide are independently activated by means of separate heterobifunctional cross-linking agents. The activated antibody and DNA label are then coupled in a single spontaneous reaction.
International patent publication no. WO 91/17442 describes a molecular probe for use as a signal amplifier in immunoassays for detecting i.a. antigens. The probe comprises an antibody, a double stranded polynucleotide functioning as a promoter for a DNA dependend RNA polymerase, and a single or double stranded template for the promoter. The transcription product is quantified and correlated to the amount of present antigen in a sample.
However, in all three of the above described immunoassays the attached DNA is only used as a marker by being amplified to detectable levels. There is no distinction between oligonucleotides attached to antibodies having bound antigen and oligonucleotides attached to antibodies not having bound antigen, i.e. those being non-specifically trapped. Non-specifically trapped antibodies give rise to an undesired background signal and limits the minimun number of antigen molecules that can be detected and it will not be possible to distinguish between false positive and true positive results below a certain number of antigen molecules. Commonly, solid supports such as microtiter plates, are used for the reactions. According to prior art, there will always be an excess of oligonucleotide-labeled antibody that cannot be removed from the solid support by adding background-lowering agents and by repeated wash steps.